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Inhibition of IGF-1 Signaling by Genistein: Modulation

of E-Cadherin Expression and Downregulation of β-
Catenin Signaling in Hormone Refractory PC-3 Prostate
Cancer Cells
a b c d a
Joomin Lee , Jihyeung Ju , Seyeon Park , Sung Joon Hong & Sun Yoon
a
Department of Food and Nutrition, Brain Korea 21 Project, Yonsei University College of
Human Ecology, Seodaemun-Gu, Seoul, Korea
b
Department of Food and Nutrition, Chungbuk National University, Chungbuk, Korea
c
Department of Applied Chemistry, Dongduk Women's University, Seoul, Korea
d
Department of Urology, Urological Science Institute, Brain Korea 21 Project for Medical
Sciences, Yonsei University College of Medicine, Seoul, Korea
Version of record first published: 18 Nov 2011.

To cite this article: Joomin Lee , Jihyeung Ju , Seyeon Park , Sung Joon Hong & Sun Yoon (2012): Inhibition of IGF-1 Signaling
by Genistein: Modulation of E-Cadherin Expression and Downregulation of β-Catenin Signaling in Hormone Refractory PC-3
Prostate Cancer Cells, Nutrition and Cancer, 64:1, 153-162

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 Nutrition and Cancer, 64(1), 153–162
Copyright
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ISSN: 0163-5581 print / 1532-7914 online

DOI: 10.1080/01635581.2012.630161

Inhibition of IGF-1 Signaling by Genistein: Modulation of

E-Cadherin Expression and Downregulation of ␤-Catenin
Signaling in Hormone Refractory PC-3 Prostate Cancer Cells
Joomin Lee
Department of Food and Nutrition, Brain Korea 21 Project, Yonsei University College of Human
Ecology, Seodaemun-Gu, Seoul, Korea

Jihyeung Ju
Department of Food and Nutrition, Chungbuk National University, Chungbuk, Korea

Seyeon Park
Downloaded by [University of Arizona] at 04:49 22 January 2013

Department of Applied Chemistry, Dongduk Women’s University, Seoul, Korea

Sung Joon Hong
Department of Urology, Urological Science Institute, Brain Korea 21 Project for Medical Sciences,
Yonsei University College of Medicine, Seoul, Korea

Sun Yoon
Department of Food and Nutrition, Brain Korea 21 Project, Yonsei University College of Human
Ecology, Seodaemun-Gu, Seoul, Korea

inhibited cell growth in IGF-1-stimulated PC-3 cells, possibly by

Elevated levels of insulin-like growth factor-1 (IGF-1) are asso-
ciated with an increased risk of several different cancers, including
prostate cancer. Inhibition of IGF-1 and the downstream signal-
ing pathways mediated by the activation of the IGF-1 receptor
(IGF-1R) may be involved in inhibiting prostate carcinogenesis. We
investigated whether genistein downregulated the IGF-1/IGF-1R
signaling pathway and inhibited cell growth in hormone refractory
PC-3 prostate cancer cells. Genistein treatment caused a significant
inhibition of IGF-1-stimulated cell growth. Flow cytometry anal-
ysis revealed that genistein significantly decreased the number of
IGF-1-stimulated cells in the G0/G1 phase of the cell cycle. In
IGF-1-treated cells, genistein effectively inhibited the phosphory-
lation of IGF-1R and the phosphorylation of its downstream tar-
gets, such as Src, Akt, and glycogen synthase kinase-3β (GSk-3β).
IGF-1 treatment decreased the levels of E-cadherin but increased
the levels of β-catenin and cyclin D1. However, genistein treat-
ment greatly attenuated IGF-1-induced β-catenin signaling that
correlated with increasing the levels of E-cadherin and decreasing
cyclin D1 levels in PC-3 cells. In addition, genistein inhibited T-
cell factor/lymphoid enhancer factor (TCF/LEF)-dependent tran-
scriptional activity. These results showed that genistein effectively
Submitted 9 May 2010; accepted in final form 25 August 2011.
Address correspondence to Sun Yoon, Department of Food and
Nutrition, Yonsei University 262 Seongsanno, Seodaemun-gu, Seoul
120-749, Korea. Phone: +82 2 2123 3119. Fax: +82 2 365 3118.
E-mail: snkim@yonsei.ac.kr
inhibiting downstream of IGF-1R activation.
INTRODUCTION
Prostate cancer is a common malignancy that is the sec-
ond leading cause of cancer-related deaths in Western males
(1). Most early prostate cancers require androgen stimulation
(androgen-dependent), and blocking androgen action through
androgen ablation is a common therapy for disease. However,
after a time, prostate cancer progresses toward a hormone re-
fractory (androgen-independent) state and there are no effective
therapeutic options for this stage of disease (2,3). Deregulation
of specific growth factors is associated with the development of
androgen ablation-refractory prostate cancer. Laboratory- and
population-based studies have linked elevated levels of insulin-
like growth factor-1 (IGF-1) to prostate cancer progression (4).
IGF-1 is postulated to contribute to prostate cancer development
by blocking apoptosis, which promotes cancer cell prolifera-
tion and invasion. The actions of IGF-1 are mediated by the
insulin-like growth factor-1 receptor (IGF-1R). IGF-1 binding
to IGF-1R allows the β-subunits of IGF-1R to display intrinsic
tyrosine kinase activity and activates downstream signals via
phosphorylation of key proteins.

153
 154 J. LEE ET AL.
IGF-1R also affects the expression or phosphorylation of dif-
ferent cell–cell adhesion proteins (5). The E-cadherin/β-catenin
complex plays an important role in maintaining proper epithelial
cell function and tissue integrity (6). IGF-1 treatment decreases
E-cadherin expression and reduces cell–cell adhesion (7). Loss
of E-cadherin expression commonly occurs when normal cells
transit to highly malignant human epithelial cancers, such
as prostate cancer (8,9).
IGF-1 induction enhances the tyrosine phosphorylation of
β-catenin. This results in the dissociation of β-catenin from
the E-cadherin complex and increase in the cytoplasmic level
of β-catenin (10). β-catenin is not only involved structurally
in the adherent junction complex but also acts as a signaling
molecule in the Wnt signaling pathway (11). The cytoplasmic
expression of β-catenin is low in normal cells because it is
easily targeted for ubiquitination by glycogen synthase kinase-
3β (GSK-3β) phosphorylation and is degraded by proteosomes
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(12,13). In cancer cells, β-catenin accumulates in the cytoplasm
and translocates into the nucleus, where it interacts with T-cell
factor/lymphoid enhancer factor (TCF/LEF) factors to activate
specific target genes, such as c-myc and cyclin D1, to promote
cancer progression.
IGF-1/IGF-1R plays a role in maintaining the malignant phe-
notype and enhanced activity of IGF-1R has been observed in
prostate cancers (14). An increase in IGF-1R expression is ob-
served in the majority of metastatic and androgen-independent
prostate cancer specimens as compared to primary disease
(15–17). IGF-1/IGF-1R pathways are related to activation of
cellular growth and proliferation signals via protein tyrosine
phosphorylation. Genistein, a soy-derived isoflavone, has re-
ceived much attention as a dietary component that acts as a
tyrosine kinase inhibitor to block or reverse carcinogenesis, in-
cluding prostate cancers (18). The present study investigated
whether genistein could overcome the growth stimulatory ef-
fects of IGF-1 in hormone refractory prostate cancer cells by in-
hibiting the activity of specific receptor tyrosine kinase (RTKs)
and related downstream pathways of signal transduction, in-
cluding the Wnt signaling pathway.
MATERIALS AND METHODS
Cell Culture
The androgen insensitive, p53-negative PC-3 human prostate
cancer cells were obtained from the Korean Cell Line
Bank (Korea). The cell line was grown in RPMI 1640
(Gibco/Invitron, Carlsbad, CA) medium supplemented with
10% heat-inactivated fetal bovine serum (FBS; Gibco), 100
units/ml penicillin, and 100 µg/ml streptomycin in an incubator
at 37◦ C with 5% CO2 .
Chemicals and Antibodies
Genistein, IGF-1, and dimethyl sulfoxide (DMSO) were
purchased from Sigma (St. Louis, MO). Propidium io-
dide (PI) was purchased from BD Pharmingen (San Diego,
CA). Genistein was dissolved in DMSO (final concentration
<0.05%). β-catenin, E-cadherin, IGF-1Rβ, phospho-IGF-1Rβ
(Tyr 1135/1136), Src, phospho-Src (Tyr 416), Akt, phospho-
Akt (Ser 473), GSK-3β, phospho-GSK-3β (Ser 9), cyclin D1,
β-actin, and histone H2A antibodies, as well as antirabbit and
antimouse peroxidase-conjugated antibodies, were purchased
from Cellular Signaling Technology (Beverly, MA). Total-ERK
and phospho-ERK (Thr202/Tyr204) antibodies were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell Viability Assay
PC-3 cells were seeded into 96-well plates at 5 × 103
cells/well. The following day, the 10% FBS medium was re-
moved, and the cells were washed once with phosphate-buffered
saline (PBS). Fresh serum-free medium with or without genis-
tein and IGF-1 (50 ng/mL) was provided. Cell viability was
determined after 24 and 48 h using the CellTiter96 Aqueous-
One-Solution Assay (Promega, Madison, WI), according to the
manufacturer’s protocol.
Cell Cycle Analysis by Flow Cytometry
Cells (5 × 105) were plated into 10-cm-diameter dishes and
allowed to adhere overnight. Cells were serum starved for 24 h
and then treated with different concentrations of genistein and
IGF-1 for an additional 24 h. Treated cells were harvested for cell
cycle analysis. Cells were washed in PBS, fixed in 70% ethanol,
and stored at −20◦ C. Before analysis, cells were washed once
with PBS and PI was added. Samples were incubated at room
temperature in the dark for 15 min. DNA content/cell number
(10,000 cells per sample) was analyzed by flow cytometry using
a FACSCalibur apparatus (Becton Dickinson, San Jose, CA).
The percentage of cells in each phase of the cell cycle was
determined by the Modfit LT program (Verity Software House,
Topsham, ME).
Preparation of Cell Extracts
Following genistein and IGF-1 treatment, the medium was
aspirated and the cells were washed with cold PBS. Total cell
extracts were prepared by scraping the cells into lysis buffer [1%
NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sul-
fate (SDS), 10 µl/ml phenylmethanesulfonyl fluoride (PMSF),
30 µl/ml aprotinin, and 10 µl/ml sodium orthovanadate]. Nu-
clear and cytoplasmic proteins were isolated using the CelLytic
NuCLEAR Extraction kit (Sigma, St. Louis, MO). Extracts were
aliqouted and stored at −80◦ C for subsequent Western blot anal-
yses. Protein concentrations were determined by the Bradford
Protein Assay (Bio-Rad, Hercules, CA).
SDS-Polyacrylamide Gel Electrophoresis
and Western Blotting
Cell extracts (15–50 µg of protein) were electrophoresed
on SDS-polyacrylamide gels and transferred to nitrocellulose
membranes. Blots were blocked in 5% nonfat dry milk/TBS
containing 0.05% Tween-20 and incubated overnight at 4◦ C
with primary antibody. The membranes were then hybridized
 GENISTEIN MODULATES β-CATENIN IN PROSTATE CANCER CELLS
with the secondary antibody conjugated to HRP for 1 h at room
temperature. Immuno reactions were detected using ECL Plus
Western blotting reagents (Amersham Pharmacia Biotech, Baie
d’Urfe, Québec, Canada). Quantitative analysis was performed
by volume densitometry after scanning the film.
Immunofluorescence Staining and Confocal
Laser Microscopy
Cells (1.5 × 105/well) were seeded onto glass coverslips,
grown for 24 h to obtain compact cell monolayers, and then
fixed in 3.7% formaldehyde for 15 min. Cells were saturated in
PBS/1% bovine serum albumin (PBS/BSA) with 0.1% Triton X-
100 and incubated with primary antibodies diluted in PBS/BSA
for 60 min at room temperature. After 3 washes in PBS, sec-
ondary fluorochrome-conjugated (Alexa Fluor 488 and Alexa
Fluor 546) antibodies in PBS/BSA were added for 60 min. Cov-
erslips were mounted on glass slides with mounting solution and
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analyzed by confocal microscopy (LSM 510 Confocal Micro-
scope, Carl Zeiss, Inc., Thornwood, NY). Nuclei were stained
with 4 ,6-diamidino-2-phenylindole (DAPI) for 5 min, and the
coverslips were mounted onto glass slides with mounting solu-
tion. Slides were analyzed by confocal microscopy (LSM 510
Confocal Microscope, Carl Zeiss, Inc.).
Transfection and Luciferase Reporter Gene Assay
Cells were transfected with reporter gene constructs using
Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to
the manufacturer’s instructions. Firefly and Renilla luciferase
activities were sequentially determined 24 h posttransfection
using the Dual Glo luciferase assay system (Promega, Madison,
WI) according to the manufacturer’s instructions.
Statistical Analyses
All the results were presented as mean values ± SD. Data
from each assay were statistically analyzed by 1-way ANOVA
with Bonferroni’s test. Differences were considered significant
when the P value was less than 0.05.
RESULTS
Genistein Decreases IGF-1-Stimulated PC-3
Cell Proliferation
We examined the effect of genistein on PC-3 cell prolifera-
tion in the presence or absence of IGF-1 stimulation using the
MTS assay. IGF-1 significantly increased PC-3 cell prolifera-
tion when added to serum-free medium (Fig. 1A). Genistein
inhibited the IGF-1 (50 ng/mL) stimulated PC-3 cell growth
in a dose- and time-dependent manner as compared to IGF-1
treatment alone (Fig. 1B). Genistein had no inhibitory effect on
PC-3 cell growth in the absence of IGF-1. The distribution of
PC-3 cells in the cell cycle was analyzed to further investigate
the mechanism of genistein-induced suppression of cell growth.
Approximately 75% of cells were blocked in G0/G1 following a
24-h serum starvation. IGF-1 stimulation significantly increased
155
the ratio of cells in S phase. There was a decrease in the per-
centage of cells in G0/G1 and an increase in the percentage
of cells in G2/M following treatment with genistein and IGF-1
(Fig. 1C).
Genistein Inhibits IGF-1-Induced Tyrosine
Phosphorylation of IGF-1R and Src
IGF-1R is a protein tyrosine kinase receptor that is involved
in the proliferation and differentiation of normal cells. IGF-
1/IGF-1R plays a key role in the growth promoting-function
of prostate cancer cells (19,20). To determine whether genis-
tein inhibited IGF-1-induced phosphorylation of IGF-1R, PC-3
cells were treated with IGF-1 (50 ng/mL) in the absence or
presence of genistein. A 30-min treatment with IGF-1 alone
showed the highest level of IGF-1R phosphorylation (Fig. 2A).
However, phosphorylation of IGF-1R decreased significantly
when the cells were cotreated with 25 or 50 µM genistein in the
presence of IGF-1 (50 ng/mL) for 30 min (Fig. 2B). We also
evaluated the effect of genistein on phosphorylation of the Src
nonreceptor protein tyrosine kinase. Src is one of the most im-
portant intracellular oncogenic signals and it participates in the
androgen-independent growth of prostate cancer cells (21–23).
IGF-1 treatment induced Src phosphorylation (Fig. 2C). A 30-
min stimulation with genistein significantly abrogated the IGF-
1-induced upregulation of Src phosphorylation. Total Src pro-
tein levels were not affected in treated cells (Fig. 2D).
Genistein Suppresses IGF-1-Induced Phosphorylation
of PI3K/Akt and GSK-3␤
IGF-1R signaling involves PI3K/Akt phosphorylation that
can promote prostate cancer cell growth (24). To investigate
whether genistein suppressed the PI3K/Akt pathway, the phos-
phorylation levels of Akt and GSK-3β were assessed. IGF-1
treatment caused an increase in Akt phosphorylation that was
the most prominent at 30 min following treatment (Fig. 3A).
However, Akt phosphorylation was significantly inhibited by
genistein in a dose-dependent manner when compared with
IGF-1 treatment alone (Fig. 3B). PC-3 cells treated for 24 h with
IGF-1 had the highest level of GSK-3β phosphorylation (Fig.
3C). This effect was inhibited by genistein in a dose-dependent
manner (Fig. 3D).
Genistein Does Not Alter IGF-1-Induced
Phosphorylation of ERK1/2
Because the ras-raf-MAPK pathway is the important down-
stream of IGF-1R, we examined the levels of phosphorylated
and total ERK1/2 by Western blots. As shown in Fig. 4A, the
ratio of phosphorylated to total ERK1/2 protein was highest fol-
lowing IGF-1 treatment for 30 min. Treatment with genistein
(1–50 µM), with or without IGF-1, did not significantly affect
the ratio of phosphorylated to total ERK1/2 protein at 30 min
(Fig. 4B).
 156 J. LEE ET AL.
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FIG. 1. Genistein inhibited insulin-like growth factor-1 (IGF-1)-induced cell proliferation in PC-3 cells. A: Cells were starved in serum-free media for 24 h and
then treated with different concentrations of IGF-1 for an additional 24 h. Number of viable cells (% of control) was measured by the MTS assay. B: Cells were
starved in serum-free media for 24 h and then treated with different concentrations of genistein for 24 h or 48 h in the absence or presence of IGF-1 (50 ng/mL).
Number of viable cells (% of control) was measured by the MTS assay. C: Cells were treated with genistein at different concentrations for 24 h in the presence of
IGF-1 (50 ng/mL). The cell cycle distribution was analyzed by flow cytometry. Data are shown as mean ± SD of triplicate determinations (n = 3). Significantly
different (P < 0.05) compared to treatment with dimethyl sulfoxide (a) and treatment with IGF-1 alone (b) by 1-way ANOVA followed by Bonferroni’s test.

Genistein Alters E-Cadherin and ␤-Catenin Levels
IGF-1R mediated signaling can result in decreased intracel-
lular adhesion due to increased tyrosine phosphorylation of E-
cadherin and β-catenin (25). Many studies have shown that the
alterations in E-cadherin/β-catenin expression are linked to dis-
ease progression via a loss of cell–cell adhesion and increased
metastasis (26–28). Therefore, we investigated the effects of
genistein and IGF-1 cotreatment on E-cadherin and β-catenin
in PC-3 cells. Cotreatment of PC-3 cells with IGF-1 and 1, 10,
25, or 50 µM of genistein for 24 h increased E-cadherin protein
levels by 104.1%, 119.4%, 162.7%, and 242.4%, respectively
(Fig. 5A) as compared to IGF-1 treatment alone. Genistein and
IGF-1 treatment also decreased the nuclear and cytoplasmic
levels of β-catenin. Reductions of 92.3%, 74.6%, 68.5%, and
33.2% in nuclear β-catenin levels and 77.4%, 60%, 47.4%,
and 40.3% in cytoplasmic levels were observed with 1, 10, 25,
and 50 µM of genistein and IGF-1 cotreatment for 24 h, respec-
tively (Fig. 5B). Fluorescence microscopy also revealed altered
 GENISTEIN MODULATES β-CATENIN IN PROSTATE CANCER CELLS 157
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FIG. 2. Genistein decreased the tyrosine phosphorylation of insulin-like growth factor-1 receptor (IGF-1R) and Src in insulin-like growth factor-1 (IGF-1)-treated
PC-3 cells. A, C: Cells were serum-starved for 24 h and then treated with 50 ng/mL IGF-1 for different times. The total and phosphorylated levels of IGF-1R
and Src were measured by Western blot analysis. Results are shown as mean fold change of 2 measurements. B, D: Cells were treated with genistein at different
concentrations for 30 min in the presence of IGF-1 (50 ng/mL). The total and phosphorylated levels of IGF-1R and Src were measured by Western blot analysis.
Results are shown as mean ± SD of 3 independent experiments (n = 3). Significantly different (P < 0.05) compared to treatment with dimethyl sulfoxide (a) and
treatment with IGF-1 alone (b) by 1-way ANOVA followed by Bonferroni’s test.

E-cadherin and β-catenin levels after IGF-1 and/or genistein
treatment (50 µM) (Fig. 5C).
Genistein Treatment Decreases ␤-Catenin-Mediated
Gene Transcription
To determine if genistein treatment could reduce β-catenin-
mediated gene transcription, cells were transiently transfected
with the TOPFlash TCF/LEF luciferase reporter construct.
Luciferase activity was significantly decreased in PC-3 prostate
cancer cells following treatment with 25 and 50 µM genistein
(Fig. 6A). Since β-catenin signaling can induce or repress the
expression of a variety of target genes, we assessed the protein
expression of cyclin D1, a transcriptional target of β-catenin,
by Western blot. Treatment of PC-3 cells with 50 µM genistein
for 24 h decreased the nuclear level of cyclin D1 by 25.5% and
 158 J. LEE ET AL.
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FIG. 3. Genistein decreased the phosphorylation of Akt and GSK-3β in insulin-like growth factor-1 (IGF-1)-treated PC-3 cells. A, C: Cells were serum-starved
for 24 h and then treated with 50 ng/mL IGF-1 for different times. Total and phosphorylated levels of Akt and glycogen synthase kinase-3β (GSK-3β) were
measured by Western blot analysis. Results are shown as mean fold change of 2 measurements. B: Cells were treated for 30 min with genistein at different
concentrations in the presence of IGF-1 (50 ng/mL). Total and phosphorylated levels of Akt were measured by Western blot analysis. D: Cells were treated for
24 h with genistein at different concentrations in the presence of IGF-1 (50 ng/mL). Total and phosphorylated levels of GSK-3β were measured by Western blot
analysis. Results are shown as mean ± SD of 3 independent experiments (n = 3) (B, D). Significantly different (P < 0.05) compared to treatment with dimethyl
sulfoxide (a) and treatment with IGF-1 alone (b) by 1-way ANOVA followed by Bonferroni’s test.

the cytoplasmic level by 60.3% as compared to IGF-1 alone
(Fig. 6B).
DISCUSSION
We showed that IGF-1 increased proliferation of PC-3
prostate cancer cells, and this increased effect was significantly
inhibited by genistein. IGF-1 stimulation increased the phos-
phorylated form of the IGF-1R protein and its downstream
signaling protein Src. The Src family of kinases (SFK) are
highly expressed in androgen-independent prostate cancer cell
lines and are responsible for multiple signal transduction events,
such as differentiation, adhesion, and migration (29). Our results
 GENISTEIN MODULATES β-CATENIN IN PROSTATE CANCER CELLS 159
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FIG. 4. Genistein did not significantly decrease ERK 1/2 phosphorylation. A: Cells were serum-starved for 24 h and then treated with 50 ng/mL insulin-like
growth factor-1 (IGF-1) for different times. Results are shown as mean fold change of 2 measurements. B: Cells were treated with different concentrations of
genistein for 30 min in the presence of IGF-1 (50 ng/mL). Total and phosphorylated levels of ERK 1/2 were measured by Western blot analysis. Results are shown
as mean ± SD of 3 independent experiments (n = 3). Significantly different (P < 0.05) compared to treatment with dimethyl sulfoxide (a) by 1-way ANOVA
followed by Bonferroni’s test.

showed that treatment of PC-3 cells with genistein inhibited the
phosphorylation of IGF-1R and Src, which were activated with
IGF-1 stimulation.
Several studies have shown that a number of RTKs and the ty-
rosine kinase Src induce tyrosine phosphorylation of β-catenin.
This releases β-catenin from the cadherin–catenin complexes
and allows it to participate in the Wnt signaling pathway (30).
Both E-cadherin and β-catenin co-localize at the surface of
epithelial cells and play crucial roles in epithelial cell–cell ad-
hesion (31). Disruption of the cadherin–catenin complex has
been linked to the invasion and metastasis of various cancer
cells, including prostate cancer (32–35).
We demonstrated that treatment of PC-3 cells with genistein
and IGF-1 significantly increased E-cadherin expression and
decreased β-catenin accumulation, as compared to treatment
with IGF-1 alone. Genistein-induced increases in E-cadherin
expression and decreases in phospho-Src levels may contribute
to the restoration of the E-cadherin/β-catenin complex. Further
studies are necessary to fully demonstrate such an association.
Since IGF-1 is one of the most potent natural activators
of the Akt signaling pathway, we investigated whether genis-
tein altered the phosphorylation status of Akt/GSK-3β in PC-3
cells stimulated with IGF-1. IGF-1 increased the phosphoryla-
tion of Akt/GSK-3β, and this effect was reversed by genistein.
Genistein-mediated dephosphorylation and activation of GSK-
3β may enhance the phosphorylation of β-catenin, leading to
its ubiquitylation and proteasome-mediated degradation. The
present results suggest that genistein may inhibit the Wnt sig-
naling pathway through inhibition of IGF-1-initiated phospho-
rylation of specific RTKs.
The Wnt signaling pathway has recently emerged as an im-
portant player in prostate tumorigenesis. Only a small percent-
age of prostate cancer samples harbor mutations in the destruc-
tion complex and β-catenin itself, suggesting that other mecha-
nisms may be involved in activating the Wnt signaling pathway
in prostate cancer progression. β-catenin, a key component of
Wnt signaling, has multifunctional roles through its associations
with various protein partners. β-catenin is involved in cell–cell
adhesion when associated with cadherins, and it regulates gene
transcription when associated with the TCF/LEF proteins. Sev-
eral studies have shown that increased nuclear localization of
β-catenin and its transcriptional activity are important events
during cancer development (33).
We demonstrated that IGF-1 increased nuclear β-catenin lev-
els and the subsequent activation of TCF/LEF transcription fac-
tor family members. IGF-1 also increased cyclin D1, one of the
target genes of the β-catenin/TCF complex, and promoted the
S cell cycle transition leading to cancer cell proliferation. How-
ever, genistein blocked IGF-1-mediated downstream signals by
dephosphorylating IGF-1R, Src, Akt, and GSK-3β and altered
E-cadherin and β-catenin levels in PC-3 prostate cancer cells.
Treatment of cells with genistein and IGF-1 decreased the per-
centage of cells in the G0/G1phases and increased the number
of cells in the G2/M phases. Animal studies have shown that
genistein treatment decreased the expression of cyclin D1 and
reduced tumor growth (36).
 160 J. LEE ET AL.
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FIG. 5. Genistein modulated the protein levels of E-cadherin and β-catenin in insulin-like growth factor-1 (IGF-1)-treated PC-3 cells. Cells were starved in
serum-free medium for 24 h and then treated with different concentrations of genistein and 50 ng/mL IGF-1 for 12 h and 24 h. A: Western blots analysis of
E-cadherin in whole cell lysates. E-cadherin levels were quantified by volume densitometry and normalized to β-actin. B: Western blot analysis of β-catenin in
cytoplasmic and nuclear fractions. Cytoplasmic and nuclear β-catenin levels (quantified by volume densitometry) were normalized to β-actin and histone H2A,
respectively. Western blot results are shown as mean ± SD of 3 independent experiments (n = 3). C: Confocal microscopic analysis of E-cadherin and β-catenin.
DAPI was used for staining the nucleus. The results are representative of 3 independent experiments that showed similar staining patterns. Significantly different
(P < 0.05) compared to treatment with dimethyl sulfoxide (a) and treatment with IGF-1 alone (b) by 1-way ANOVA followed by Bonferroni’s test.
 GENISTEIN MODULATES β-CATENIN IN PROSTATE CANCER CELLS 161
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FIG. 6. Genistein inhibited β-catenin-mediated gene transcription and cyclin D1 expression in insulin-like growth factor-1 (IGF-1)-treated PC-3 cells. A:
Cells were transiently transfected with TOPflash and FOPflash reporter gene constructs. Twenty-four hours after transfection, cells were treated with different
concentrations of genistein and IGF-1 (50 ng/mL). B: Nontransfected cells were starved in serum-free medium for 24 h and then treated with 50 µM genistein
for an additional 24 h in the presence of 50 ng/mL IGF-1. Nuclear and cytosolic cyclin D1 levels were measured by Western blot analysis and normalized to
histone H2A and β-actin, respectively. Nuclear protein, as represented by histone H2A, was not detected in the cytoplasmic fractions. This indicates that there
was no significant cross-contamination between nuclear and cytoplasmic fractions in our preparation. Representative results of 3 independent experiments are
shown. Significantly different (P < 0.05) compared to treatment with dimethyl sulfoxide (a) and treatment with IGF-1 alone (b) by 1-way ANOVA followed by
Bonferroni’s test.

After androgen deprivation at late stage in prostate cancer,
tumor of castration-resistant prostate cancer (CRPC) continues
to depend on androgen receptor (AR). IGF-1/IGF-1R is one
of the key signaling pathways that activate AR (37). Our stud-
ies in PC-3 cells showed that, in absence of AR, IGF-1 al-
ternatively modulated E-cadherin and β-catenin pathways and
genistein decreased IGF-1/IGF-1R mediated downstream sig-
nals. Further studies are needed to consider AR and E-cadherin
/β-catenin pathways with genistein treatment. Genistein plasma
levels in people on a soy-rich diet are in the 1–5 µM range af-
ter metabolism and excretion (38,39). At low doses (<10 µM),
genistein stimulated the growth of estrogen-sensitive cell lines
(40), while decreasing proliferation at higher doses (>10–20
µM) (41). This suggests that genistein exerts a biphasic effect
on growth and proliferation of cancer cells. Genistein has multi-
ple biochemical effects and can act as an antiproliferative agent
at high concentrations (≥10 µM).
In conclusion, genistein exerts its role as a critical inhibitor
of RTKs through a series of signal transduction events. Genis-
tein also increases E-cadherin and decreases β-catenin, which
results in inhibition of TCF/LEF transcription factor binding to
Wnt target gene promoters in PC-3 prostate cancer cells. These
observations indicate that genistein may play a role in the pre-
vention and/or treatment of hormone refractory prostate cancer.
ACKNOWLEDGMENTS
This study was supported by a grant of the Korea Health
21 R&D project, Ministry of Health and Welfare, Republic of
Korea (Contact grant number A050611).
REFERENCES
1. Debes JD and Tindall DJ: Mechanisms of androgen-refractory prostate
cancer. N Engl J Med 351, 1488–1490, 2004.
2. Schröder FH: Progress in understanding androgen-independent prostate
cancer (AIPC): a review of potential endocrine-mediated mechanisms. Eur
Urol 53, 1129–1137, 2008.
 162 J. LEE ET AL.
3. Miyamoto H, Messing EM, and Chang C: Androgen deprivation therapy for
prostate cancer: current status and future prospects. Prostate 61, 332–353,
2004.
4. Renehan AG, Zwahlen M, Minder C, O’Dwyer ST, Shalet SM, et al.:
Insulin-like growth factor (IGF)-I, IGF binding protein-3, and cancer risk:
systematic review and meta-regression analysis. Lancet 363, 346–353,
2004.
5. Mauro L, Salerno M, Morelli C, Boterberg T, Bracke ME, et al.: Role of
the IGF-I receptor in the regulation of cell–cell adhesion: implications in
cancer development and progression. J Cell Physiol 194, 108–116, 2003.
6. Nelson WJ and Nusse R: Convergence of Wnt, beta-catenin, and cadherin
pathways. Science 5, 1483–1487, 2004.
7. Ozawa M and Kemler R: Altered cell adhesion activity by pervanadate due
to the dissociation of alpha-catenin from the E-cadherin.catenin complex.
J Biol Chem 273, 6166–6170, 1998.
8. Christofori G and Semb H: The role of the cell-adhesion molecule E-
cadherin as a tumour-suppressor gene. Trends Biochem Sci 24, 73–76,
1999.
9. Paul R, Ewing CM, Jarrard DF, and Isaacs WB: The cadherin cell–cell
adhesion pathway in prostate cancer progression. Br J Urol 79(Suppl 1),
Downloaded by [University of Arizona] at 04:49 22 January 2013
37–43, 1997.
10. Playford MP, Bicknell D, Bodmer WF, and Macaulay VM: Insulin-like
growth factor 1 regulates the location, stability, and transcriptional activity
of beta-catenin. Proc Natl Acad Sci USA 97, 12103–12108, 2000.
11. MacDonald BT, Tamai K, and He X: Wnt/beta-catenin signaling: compo-
nents, mechanisms, and diseases. Dev Cell 17, 9–26, 2009.
12. Aberle H, Bauer A, Stappert J, Kispert A, and Kemler R: Beta-catenin is
a target for the ubiquitin-proteasome pathway. EMBO J 16, 3797–3804,
1997.
13. Yost C, Torres M, Miller JR, Huang E, Kimelman D, et al.: The axis-
inducing activity, stability, and subcellular distribution of beta-catenin is
regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev
10, 1443–1454, 1996.
14. Pollak MN, Schernhammer ES, and Hankinson SE: Insulin-like growth
factors and neoplasia. Nat Rev Cancer 4, 505–518, 2004.
15. Hellawell GO, Turner GD, Davies DR, Poulsom R, Brewster SF, et al.:
Expression of the type 1 insulin-like growth factor receptor is upregulated
in primary prostate cancer and commonly persists in metastatic disease.
Cancer Res 62, 2942–2950, 2002.
16. Krueckl SL, Sikes RA, Edlund NM, Bell RH, Hurtado-Coll A, et al.:
Increased insulin-like growth factor I receptor expression and signaling
are components of androgen-independent progression in a lineage-derived
prostate cancer progression model. Cancer Res 64, 8620–8629, 2004.
17. Nickerson T, Chang F, Lorimer D, Smeekens SP, Sawyers CL, et al.: In
vivo progression of LAPC-9 and LNCaP prostate cancer models to androgen
independence is associated with increased expression of insulin-like growth
factor I (IGF-I) and IGF-I receptor (IGF-IR). Cancer Res 61, 6276–6280,
2001.
18. Perabo FG, Von Low EC, Ellinger J, von Rucker A, Muller SC, et al.: Soy
isoflavone genistein in prevention and treatment of prostate cancer. Prostat
Cancer Prostatic Dis 11, 6–12, 2008.
19. Baserga R: The insulin-like growth factor I receptor: a key to tumor growth?
Cancer Res 55, 249–252, 1995.
20. Pandini G, Mineo R, Frasca F, Roberts CT, Jr., Marcelli M, et al.: Androgens
upregulate the insulin-like growth factor-I receptor in prostate cancer cells.
Cancer Res 65, 1849–1857, 2005.
21. Lee LF, Guan J, Qiu Y, and Kung HJ: Neuropeptide-induced androgen
independence in prostate cancer cells: roles of nonreceptor tyrosine kinases
Etk/Bmx, Src, and focal adhesion kinase. Mol Cell Biol 21, 8385–8397,
2001.
22. Lee LF, Louie MC, Desai SJ, Yang J, Chen HW, et al.: Interleukin-8 confers
androgen-independent growth and migration of LNCaP: differential effects
of tyrosine kinases Src and FAK. Oncogene 23, 2197–2205, 2004.
23. Unni E, Sun S, Nan B, McPhaul MJ, Cheskis B, et al.: Changes in androgen
receptor nongenotropic signaling correlate with transition of LNCaP cells
to androgen independence. Cancer Res 64, 7156–7168, 2004.
24. Grimberg A: Mechanisms by which IGF-I may promote cancer. Cancer
Biol Ther 2, 630–635, 2003.
25. Bahr C and Groner B: The IGF-1 receptor and its contributions to metastatic
tumor growth-novel approaches to the inhibition of IGF-1R function.
Growth Factors 23, 1–14, 2005.
26. Jaggi M, Johansson SL, Baker JJ, Smith LM, Galich A, et al.: Aberrant
expression of E-cadherin and beta-catenin in human prostate cancer. Urol
Oncol 23, 402–406, 2005.
27. Morita N, Uemura H, Tsumatani K, Cho M, Hirao Y, et al.: E-cadherin and
alpha-, beta-, and gamma-catenin expression in prostate cancers: correlation
with tumour invasion. Br J Cancer 79, 1879–1883, 1999.
28. Patriarca C, Petrella D, Campo B, Colombo P, Giunta P, et al.: Ele-
vated E-cadherin and alpha/beta-catenin expression after androgen depri-
vation therapy in prostate adenocarcinoma. Pathol Res Pract 199, 659–665,
2003.
29. Nam S, Kim D, Cheng JQ, Zhang S, Lee JH, et al.: Action of the Src family
kinase inhibitor, dasatinib (BMS-354825), on human prostate cancer cells.
Cancer Res 65, 9185–9199, 2005.
30. Lilien J, Balsamo J, Arregui C, and Xu G: Turn-off, drop-out: functional
state switching of cadherins. Dev Dyn 224, 18–29, 2002.
31. Jamora C and Fuchs E: Intercellular adhesion, signaling and the cytoskele-
ton. Nat Cell Biol 4, E101–E108, 2002.
32. Chen HC, Chu RY, Hsu PN, Hsu PI, Lu JY, et al.: Loss of E-cadherin
expression correlates with poor differentiation and invasion into adjacent
organs in gastric adenocarcinomas. Cancer Lett 201, 97–106, 2003.
33. Chesire DR and Isaacs WB: Beta-catenin signaling in prostate cancer: an
early perspective. Endocr Relat Cancer 10, 537–560, 2003.
34. Umbas R, Schalken JA, Aalders TW, Carter BS, Karthaus HF, et al.: Ex-
pression of the cellular adhesion molecule E-cadherin is reduced or absent
in high-grade prostate cancer. Cancer Res 52, 5104–5109, 1992.
35. Verras M and Sun Z: Roles and regulation of Wnt signaling and beta-catenin
in prostate cancer. Cancer Lett 237, 22–32, 2006.
36. El Touny LH and Banerjee PP: Akt–GSK-3 pathway as a target in
genistein-induced inhibition of TRAMP prostate cancer progression to-
ward a poorly differentiated phenotype. Carcinogenesis 28, 1710–1717,
2007.
37. Wang Y, Kreisberg JI, and Ghosh PM: Cross-talk between the androgen
receptor and the phosphatidylinositol 3-kinase/Akt pathway in prostate
cancer. Curr Cancer Drug Targets 7, 591–604, 2007.
38. Adlercreutz H, Markkanen H, and Watanabe S: Plasma concentrations of
phyto-oestrogens in Japanese men. Lancet 13, 1209–1210, 1993.
39. Uehar M, Arai Y, Watanabe S, and Adlercreutz H: Comparison of plasma
and urinary phytoestrogens in Japanese and Finnish women by time-
resolved fluoroimmunoassay. Biofactors 12, 217–225, 2000.
40. Limer JL, Parkes AT, and Speirs V: Differential response to phytoestrogens
in endocrine sensitive and resistant breast cancer cells in vitro. Int J Cancer
119, 515–521, 2006.
41. Moore AB, Castro L, Yu L, Zheng X, Di X, et al.: Stimulatory and inhibitory
effects of genistein on human uterine leiomyoma cell proliferation are
influenced by the concentration. Hum Reprod 22, 2623–2631, 2007.